Placentation requires faithful allocation and differentiation of trophoblast stem cells into diverse cell types, creating an organ capable of sustaining the fetus. Defects in this process can have dire consequences for both the fetus and the mother and can contribute to infertility, recurrent miscarriages and stillbirth. Abnormal fetal growth caused by placental defects can also have chronic effects throughout life. This proposal will generate insights into the epigenetic events that control human trophoblast differentiation essential for understanding the origin and pathology of placental defects. Genetic studies in mice have identified regulators of trophoblast differentiation but their clinical relevance is uncertain due to differences between mice and humans. The derivation of human trophoblast stem (hTS) cells, which are able to recapitulate early trophoblast differentiation in vitro, represents a transformational advance. This culture system provides an incisive means to investigate the effects of genetic mutations in human trophoblasts. These studies will use hTS cells to ask whether regulators identified in the mouse are crucial for human trophoblast differentiation. The long-term goal of the proposed work is to identify genetic and epigenetic factors that promote normal placental development in humans. This application focuses on BCOR, an X-linked gene with essential roles in placental development in mice. BCOR is mutated in human syndromes involving low birth weight and intrauterine growth restriction (IUGR) and women with a heterozygous null mutation in BCOR can experience recurrent miscarriages. BCOR is a conserved component of the epigenetic silencing complex Polycomb Repressive Complex 1.1. The central hypothesis of this proposal is that BCOR is a key epigenetic regulator of human placental development. The proposed work has two specific aims. The first investigates whether BCOR regulates human trophoblast differentiation by introducing into hTS cells BCOR null mutations found in human patients that might be responsible for the associated IUGR and recurrent miscarriages. Phenotypic assays will determine whether these mutations affect the ability of trophoblast stem cells to differentiate into syncytiotrophoblast and extravillous trophoblasts cell types, which play important functional roles in placentation. BCOR-dependent mRNA expression and epigenetic changes will be used to identify key downstream targets that affect trophoblast differentiation and might encode useful biomarkers for early diagnosis of pregnancy complications arising from improper trophoblast differentiation. The second aim investigates the functional importance of BCOR missense mutations, asking how they affect function of the protein. Characterization of missense mutations in the BCOR protein will provide mechanistic insights into how it influences trophoblast differentiation and will have an impact that extends beyond the patients with these syndromes.